Supercritical fluids have unique properties, since they combine liquid-like solvent power with gas-like transport properties. They have a large compressibility compared to ideal gases. Therefore, a small change in temperature or pressure near the critical values will result in large changes in the fluid's density and hence its solvent power. These characteristics can be utilised to provide highly controllable solvation properties.
Carbon dioxide is the most widely used supercritical fluid, due to the favourable critical parameters (Tc=31.1° C., Pc=73.8 bar), cost and non-toxicity.
Two principles for precipitating particles with supercritical fluids have been developed, Rapid Expansion of Supercritical Solutions (RESS) and Supercritical Anti-solvent (SAS) or Gas Anti-solvent (GAS) precipitation.
In the RESS process the sensitivity of solvent power of a supercritical fluid to small changes in pressure is used to trigger a mechanical precipitation of particles. However RESS is not suitable for use with substances such as peptides and proteins that have low solubility in the supercritical fluid.
The SAS or GAS processes can be used to precipitate particles of a substance that is insoluble in the supercritical fluid, provided that the supercritical fluid is miscible with the liquid in which the substance is dissolved.
WO 95/01221, WO 96/00610, WO 98/36825, WO 99/44733, WO 99/59710, WO 01/03821 and WO 01/15664 describe Solution Enhanced Dispersion by Supercritical fluids (SEDS) processes, which in addition to the anti-solvent properties of the supercritical fluid, has coupled a physicomechanical function. The SEDS technique involves a continuous flow of a solution containing the substance to be precipitated and the supercritical fluid, co-introduced through a coaxial nozzle into a particle-formation vessel which leads to substantially simultaneous dispersion and mixing of the solution, rapid supersaturation and particle nucleation and formation. By varying the process conditions used in the SEDS process the properties of the resulting particles can be controlled.
Precipitation of proteins using supercritical carbon dioxide has to date mainly been limited to dissolving the proteins in organic solvents such as dimethylsulfoxide (DMSO) and N,N-dimethylformamide (DMF). However, the use of such solvents to dissolve the protein can result in unfolding or denaturing of the protein. This can give rise to a loss in therapeutic effect (Winters et al, Precipitation of Proteins in Supercritical Carbon Dioxide, 1996, J. Pharm Sci, 85, 586-594, Jackson et al, Beware of Proteins in DMSO, Biochem et Biophysica Acta 1078, 231-235).
Furthermore, the rapid extraction of the organic solvent by the supercritical fluid tends to promote agglomeration of the particles as they precipitate from the solution due to the rapid nucleation and particle growth.
The precipitation of proteins from an aqueous solution would avoid damage to the protein caused by the presence of organic solvents such as DMSO. However the poor solubility of supercritical carbon dioxide in aqueous solutions and vice versa is a major hindrance to using anti-solvent techniques such as SEDS™ with aqueous solutions of many substances such as proteins. The use of anti-solvent techniques such as SEDS™ to precipitate particles from an aqueous solution results in the formation of large particles compared to precipitation from solvents such as DMSO. The formation of large particles is undesirable when, for example, the precipitated particles will be used as a pulmonary medicament which is inhaled by patients. In such cases the particles preferably have a particle diameter of less than 10 microns.
WO 96/00610 discloses the preparation of protein particles from a solution of the protein in water and ethanol using the SEDS technique. In WO 96/00610 a three-channelled co-axial nozzle is used to mix an aqueous solution of the protein with the ethanol just prior to dispersion in supercritical carbon dioxide. It is thought that the presence of ethanol improves the solubility match between carbon dioxide and water and thereby improves the efficiency of the SEDS process.
WO 99/52507 describes a process for incorporating an active substance in a carrier matrix wherein a stable water-in-oil emulsion is prepared comprising a continuous non-aqueous phase and a discontinuous aqueous phase, the active substance being present in the discontinuous aqueous phase and the carrier being present in either the aqueous phase or the non-aqueous phase. Particles comprising the active substance and carrier are then formed by contacting the stable water-in-oil emulsion with an antisolvent fluid gas using, for example the SEDS technique.
WO 97/14407 describes a process for the preparation of water-insoluble drug particles having an average particle size of from 100 nm to 300 nm by spraying a solution of the drug in an organic solvent into a compressed gas, liquid or super-critical fluid containing a surface modifier dissolved in an aqueous phase. The presence of the aqueous phase is stated to reduce agglomeration of the precipitated particles.
U.S. Pat. No. 6,299,906 describes a process in which a drug is dissolved in compressed dimethylether optionally containing a surface modifier and the resulting solution is sprayed into an anti-solvent.
Although the use of water as a solvent is desirable for many substances such as proteins, traces of water in the precipitated particles can result in the formation of tightly bound particles agglomerates. This is undesirable as such agglomerates are difficult to break up and, in effect, give rise to a large particle size distribution. The formation of tightly bound agglomerates is a particular disadvantage when the particulate product is designed to be used as an inhalable medicament. In these applications the formation of small (preferably sub-micron) particles of a uniform size are required to ensure an accurate dose of the medicament. Thus any particle agglomerates which do form should be loosely bound so that they can be easily broken up prior to administration to a patient (for example through the use of an inhaler). Furthermore, the agglomeration of precipitated particles can cause blockages in the filtration systems used in the SEDS apparatus giving rise to over-pressurisation of the apparatus. The presence of such agglomerates therefore precludes the development of a continuous/extended precipitation process.
The poor solubility of water in super critical fluids such as carbon dioxide also results in a relatively slow extraction of water and therefore a slow precipitation of particles. Such conditions favour the formation of large (>1 μm) particles.
There is therefore a need for a process which enables the formation of particles (especially sub-micron particles of a uniform size) from an aqueous medium which avoids some or all of the difficulties described above.